Escherichia coli cells defective for the recN gene display constitutive elevation of mutagenesis at 3,N(4)-ethenocytosine via an SOS-induced mechanism.

The Escherichia coli UVM (UV Modulation of mutagenesis) response is a DNA damage-inducible mutagenic pathway detected as significantly increased mutagenesis at 3,N4-ethenocytosine (epsilon C) lesions borne on transfected single-stranded M13 vector DNA. All major classes of DNA-damaging agents can induce UVM, and the phenomenon is independent of previously characterized mutagenic responses in E. coli. To understand this phenomenon further, we set out to identify and characterize mutants in the UVM response. Screening a mutant bank of cells defective for 1-methyl-3-nitro-1-nitrosoguanidine-inducible genes revealed that defects in the recN gene cause a constitutive elevation of mutagenesis at epsilon C residues. In contrast to normal cells that show approximately 6% mutagenesis at epsilon C lesions, but approximately 60% upon UVM induction, recN-defective strains display approximately 50% mutagenesis at epsilon C lesion sites in untreated cells. However, the recN-mediated mutagenesis response was found to require the recA gene and the umuDC genes, and could be suppressed in the presence of a plasmid harbouring the SOS transcriptional repressor LexA. These results imply that recN cells are constitutively active for SOS mutagenesis functions. The observation that epsilonC mutagenesis is enhanced in recN cells confirms previous findings that mutagenesis at epsilonC can also be independently elevated by the SOS pathway.

Role of mismatch repair in the Escherichia coli UVM response.

Mutagenesis at 3,N4-ethenocytosine (epsilonC), a nonpairing mutagenic lesion, is significantly enhanced in Escherichia coli cells pretreated with UV, alkylating agents, or H2O2. This effect, termed UVM (for UV modulation of mutagenesis), is distinct from known DNA damage-inducible responses, such as the SOS response, the adaptive response to alkylating agents, or the oxyR-mediated response to oxidative agents. Here, we have addressed the hypothesis that UVM results from transient depletion of a mismatch repair activity that normally acts to reduce mutagenesis. To test whether the loss of mismatch repair activities results in the predicted constitutive UVM phenotype, E. coli cells defective for methyl-directed mismatch repair, for very-short-patch repair, or for the N-glycosylase activities MutY and MutM were treated with the UVM-inducing agent 1-methyl-3-nitro-1-nitrosoguanidine, with subsequent transfection of M13 viral single-stranded DNA bearing a site-specific epsilonC lesion. Survival of the M13 DNA was measured as transfection efficiency, and mutation fixation at the lesion was characterized by multiplex sequencing technology. The results showed normal UVM induction patterns in all the repair-defective strains tested. In addition, normal UVM induction was observed in cells overexpressing MutH, MutL, or MutS. All strains displayed UVM reactivation, the term used to describe the increased survival of epsilonC-containing DNA in UVM-induced cells. Taken together, these results indicate that the UVM response is independent of known mismatch repair systems in E. coli and may thus represent a previously unrecognized misrepair or misreplication pathway.

Functional recA, lexA, umuD, umuC, polA, and polB genes are not required for the Escherichia coli UVM response.

The Escherichia coli UVM response is a recently described phenomenon in which pretreatment of cells with DNA-damaging agents such as UV or alkylating agents significantly enhances mutation fixation at a model mutagenic lesion (3,N4-ethenocytosine; epsilon C) borne on a transfected M13 single-stranded DNA genome. Since UVM is observed in delta recA cells in which SOS induction should not occur, UVM may represent a novel, SOS-independent, inducible response. Here, we have addressed two specific hypothetical mechanisms for UVM: (i) UVM results from a recA-independent pathway for the induction of SOS genes thought to play a role in induced mutagenesis, and (ii) UVM results from a polymerase switch in which M13 replication in treated cells is carried out by DNA polymerase I (or DNA polymerase II) instead of DNA polymerase III. To address these hypotheses, E. coli cells with known defects in recA, lexA, umuDC, polA, or polB were treated with UV or 1-methyl-3-nitro-1-nitrosoguanidine before transfection of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion. Survival of the transfected DNA was measured as transfection efficiency, and mutagenesis at the epsilon C residue was analyzed by a quantitative multiplex DNA sequencing technology. Our results show that UVM is observable in delta recA cells, in lexA3 (noninducible SOS repressor) cells, in LexA-overproducing cells, and in delta umuDC cells. Furthermore, our data show that UVM induction occurs in the absence of detectable induction of dinD, an SOS gene. These results make it unlikely that UVM results from a recA-independent alternative induction pathway for SOS gene.

Alkylating agents induce UVM, a recA-independent inducible mutagenic phenomenon in Escherichia coli.

Noninstructive DNA damage in Escherichia coli induces SOS functions hypothesized to be required for mutagenesis and translesion DNA synthesis at noncoding DNA lesions. We have recently demonstrated that in E. coli cells incapable of SOS induction, prior UV-irradiation nevertheless strongly enhances mutagenesis at a noninstructive lesion borne on M13 DNA. Here, we address the question whether this effect, named UVM for UV modulation of mutagenesis, can be induced by other DNA damaging agents. Exponentially growing delta recA cells were pretreated with alkylating agents before transfection with M13 single-stranded DNA bearing a site-specific ethenocytosine residue. Effect of cell pretreatment on survival of the transfected DNA was determined as transfection efficiency. Mutagenesis at the ethenocytosine site in pretreated or untreated cells was analyzed by multiplex DNA sequencing, a phenotype-independent technology. Our data show that 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurea and dimethylsulfate, but not methyl iodide, are potent inducers of UVM. Because alkylating agents induce the adaptive response to defend against DNA alkylation, we asked if the genes constituting the adaptive response are required for UVM. Our data show that MNNG induction of UVM is independent of ada, alkA and alkB genes and define UVM as an inducible mutagenic phenomenon distinct from the E. coli adaptive and SOS responses.

UVM, an ultraviolet-inducible RecA-independent mutagenic phenomenon in Escherichia coli.

Most mutagenic DNA lesions are noninstructive in the sense that template instruction is either missing or inaccessible during DNA replication, leading to replication arrest. According to the SOS hypothesis, arrested replication induces the expression of SOS factors that force replication past stalled sites at the cost of mutagenesis. We have recently shown that prior UV irradiation of delta recA cells, in which the SOS pathway does not function, enhances mutagenesis at an ethenocytosine residue borne on a circular gapped duplex DNA vector, indicating the existence of an SOS-independent inducible mutagenic phenomenon termed UVM (UV modulation of mutagenesis). In the previous experiments, mutation fixation was expected to occur during gap-filling DNA synthesis. To test whether UVM is observable during normal replication by DNA polymerase III, we have examined mutagenesis at an epsilon C residue borne on M13 single-stranded DNA. By analyzing mutation frequency and specificity using a multiplex sequence assay, we now show that UVM is observable in UV-irradiated recA+, and in delta recA cells. These data indicate that UV irradiation induces a previously unrecognized mutagenic mechanism in Escherichia coli, and that this mechanism is manifested during gap-filling DNA synthesis as well as during normal DNA replication.

Base incorporation and extension at a site-specific ethenocytosine by Escherichia coli DNA polymerase I Klenow fragment.

Ethenocytosine (epsilon C) is a highly mutagenic exocyclic DNA lesion induced by carcinogens vinyl chloride and urethane. We have examined base incorporation and extension at a site-specific epsilon C residue by a quantitative gel electrophoretic assay using an exonuclease-deficient version of Escherichia coli DNA polymerase I (Klenow fragment) as the model enzyme. The data show that the KM for incorporation of adenine or thymine opposite epsilon C by is about 5 orders of magnitude higher than that for the incorporation of guanine opposite normal cytosine. The KM for base extension past epsilon C:A and epsilon C:T pairs is 1-2 orders of magnitude higher than that observed for a C:G pair. Although adenine misinsertion is favored over that of thymine, base extension occurs more readily when the base incorporated opposite epsilon C is thymine.

Quantitative multiplex sequence analysis of mutational hot spots. Frequency and specificity of mutations induced by a site-specific ethenocytosine in M13 viral DNA.

We describe an assay for determining the frequency and specificity of mutations occurring at hot spots within a population of DNA molecules. The procedure consists of (a) annealing the DNA population with a labeled oligonucleotide designed to prime DNA synthesis at the mutational hot spot; (b) DNA elongation in the presence of a single dideoxynucleoside triphosphate together with 1-3 deoxynucleoside triphosphates, and (c) quantitation of all limit elongation products by high-resolution gel electrophoresis followed by autoradiography and computing densitometry. Derivation of mutational frequency and specificity over a wide range of values is demonstrated for M13 viral DNA mixtures containing defined proportions of wild-type and mutant DNAs, as well as for M13 viral DNA populations obtained by transfection of DNA bearing a defined site-specific ethenocytosine lesion. The assay is shown to yield results similar to those obtained by laborious clone-by-clone sequencing of viral progeny. The method is not affected significantly by several tested variables and appears to be suitable for use as a quantitative assay for sequence microheterogeneity at defined positions within DNA populations. Application of the methodology demonstrates that ethenocytosine, an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly efficient mutagenic lesion with a mutational specificity expected for noninstructive lesions.

UV irradiation of Escherichia coli modulates mutagenesis at a site-specific ethenocytosine residue on M13 DNA. Evidence for an inducible recA-independent effect.

Mutagenic action of chemical and physical mutagens is mediated through DNA damage and subsequent misreplication at sites of unrepaired damage. Most DNA damage is noninstructive in the sense that the causative chemical modification either destroys the template information or renders it inaccessible to the DNA polymerase. Noninstructive adducts possess high genotoxicity because they stop DNA replication. Replication past noninstructive adducts is thought to depend on induced functions in addition to the regular replication machinery. In Escherichia coli, noninstructive DNA damage leads to induction of the SOS regulon, which in turn is thought to provide the inducible functions required for replicative bypass of the lesion. Because of the absence of accessible template instruction, base incorporation opposite noninstructive lesions is inherently error-prone and results in mutagenesis. Ethenocytosine (epsilon C), an exocyclic DNA lesion induced by carcinogens such as vinyl chloride and urethane, is a highly mutagenic, noninstructive lesion on the basis of its template characteristics in vivo and in vitro. However, mutagenesis at epsilon C does not require SOS functions, as evidenced by efficient mutagenesis in recA-deleted E. coli. Even though efficient mutagenesis in recA-deleted cells shows a lack of SOS dependence, the question remains whether SOS induction can modulate mutagenesis opposite epsilon C. To examine the possible contribution of SOS functions to mutagenesis at epsilon C, we constructed an M13 duplex circular DNA molecule containing an epsilon C residue at a unique site. The construct was transfected into nonirradiated or UV-irradiated E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of mutagenesis by exocyclic DNA adducts. Construction and in vitro template characteristics of an oligonucleotide bearing a single site-specific ethenocytosine.

By using a gene-targeted random DNA adduction approach, we have recently shown that chloroacetaldehyde, a metabolite of vinyl chloride, induces mutations predominantly at cytosines under conditions in which both ethenoadenine (epsilon A) and ethenocytosine (epsilon C) are formed. Although the observed mutational specificity of epsilon C suggested that it was a noninstructional lesion, the high efficiency of mutagenesis and an apparent lack of SOS dependence were reminiscent of mispairing lesions. To obtain more direct evidence showing that epsilon C has properties of a noninstructional mutagenic lesion, we have examined the in vitro template properties of a single epsilon C residue at a unique position in a synthetic oligonucleotide. The oligonucleotide was constructed by use of the following steps: (a) in vitro treatment of the pentameric oligodeoxyribonucleotide TTCTT with chloroacetaldehyde to convert the central cytosine to ethenocytosine; (b) purification and characterization of TT epsilon CTT; and (c) ligation of purified TT epsilon CTT to two decamers to create a 25 nt long oligodeoxyribonucleotide with a centrally located epsilon C residue. The template characteristics of epsilon C were examined by the annealing of end-labeled primers to the purified epsilon C-containing oligonucleotide and primer elongation by Escherichia coli DNA polymerase I in the presence of one or more nucleotide precursors. The elongation products were analyzed by high-resolution gel electrophoresis followed by autoradiography and quantitated by computing densitometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient RecA-independent mutagenic noninstructional lesion.

It is widely accepted that mutagenic DNA lesions fall into two categories: mispairing lesions hydrogen bond with an incorrect incoming base, generally do not stop replication, and possess high mutagenic efficiency without any requirement for induced functions; noninstructional lesions lack accessible template information, act as strong blocks to DNA replication (and are therefore toxic), and their mutagenic effects are SOS-dependent. Our recent results show that ethenocytosine (epsilon C), a noninstructional exocyclic DNA lesion induced by vinyl chloride, may have unusual mutagenic properties. To obtain more definitive experimental evidence for the observed effects, we have introduced a single epsilon C residue at a specific site of coliphage M13AB28 replicative form DNA by a "single-stranded linker-ligation" technique. The resulting DNA was purified and transfected into appropriate recA+ or recA- Escherichia coli host cells. The effect of epsilon C on survival was determined from transfection efficiency. Both the frequency and specificity of mutations induced by epsilon C were determined by direct sequence analysis of randomly picked progeny phage plaques. The results indicated that epsilon C has little effect on the survival of M13 DNA. Approximately 30% of the progeny phage obtained by transfecting epsilon C DNA had a base substitution mutation precisely at the lesion site. No such mutations were observed in progeny plaques obtained by transfecting the control DNA construct. All epsilon C-induced mutations were either C-to-T transitions or C-to-A transversions. Neither survival nor mutagenic efficiency was significantly affected in recA- host cells.(ABSTRACT TRUNCATED AT 250 WORDS)